1. Field of the Invention
Thyroxine is an important hormone in the mammalian physiology, being excreted by the thyroid gland. The measurement of thyroid hormones such as thyroxine is an important diagnostic tool in the determination of disease such as hypothyroidism and hyperthyroidism. Furthermore, monitoring of thyroid hormone levels in patients receiving therapy for thyroid disease is commonly done. Various techniques have been used for the determination of thyroid hormones, including radioimmunoassay, competitive protein binding, chromatography, etc. These techniques suffer from a number of disadvantages in being difficult to carry out and in the case of radioimmunoassay having unstable reagents. Enzyme immunoassays such as homogeneous enzyme immunoassays have also been utilized for the determination of total thyroxine and serum thyroid protein binding capacity.
Use of complete thyroxine molecules as part of enzyme conjugates, such as conjugates of the complete thyroxine molecule with glucose-6-phosphate dehydrogenase (G-6-PDH), for competitive homogeneous enzyme immunoassays has resulted in several difficulties. In the first place the binding of antibody to the conjugates does not produce the requisite inhibition of enzyme activity necessary to achieve an adequate change of enzyme activity so that a sensitive assay is achieved. Secondly, the complete thyroxine molecule is bulky and has hydrophobic properties, which serve to destabilize an enzyme to which it is attached. In addition, G-6-PDH conjugates containing complete thyroxine molecules are very sensitive to binding of sample serum proteins, such as immunoglobulins, and are deactivated by such binding of serum proteins when it occurs in a certain small percentage of patient samples. The sample serum protein binding interference is inhibitory to the enzyme conjugate and negatively affects the performance of the immunoassay in terms of quantitation of the thyroid hormone analyte. The exact mechanism of the protein binding in such samples is not clear, but it may be a manifestation of autoimmune disorders in which antibodies to thyroxine-like molecules are present.
2. Description of the Related Art
U.S. Pat. No. 3,817,837 describes enzyme immunoassays. U.S. Pat. No. 4,040,907 discloses iodothyronine enzyme conjugates. U.S. Pat. No. 4,171,244 discloses enzyme-bound-polyiodothyronine. A polyiodothyronine immunoassay is described in U.S. Pat. No. 4,043,872. U.S. Pat. No. 4,121,975 teaches a pretreatment of samples for polyiodothyronine assays. Enzyme immunoassays with glucose-6-phosphate dehydrogenase are described in U.S. Pat. No. 3,875,011. A method for the measurement of free thyroxine or 3,5,3'-triiodothyronine in a liquid sample is described in U.S. Pat. No. 4,410,633. Iodothyronine immunogens and antibodies are taught in U.S. Pat. No. 4,399,121. A thyroxine radioimmunoassay is described in U.S. Pat. No. 4,018,883. A radioimmunoassay for measurement of thyroxine and triiodothyronine in blood serum is disclosed in U.S. Pat. No. 3,911,096. A radioimmunoassay method for triiodothyronine and thyroxine is taught in U.S. Pat. No. 3,928,553. U.S. Pat. No. 4,847,195 discloses glucose-6 -phosphate dehydrogenase conjugates useful in polyiodothyronine assays.